Results of MPSS analysis of Alexandrium fundyense cells grown under N- or P-starvation. Each data point represents a single signature sequence whose expression is significantly different (p<0.01) under the two conditions. Expression levels are given in transcripts per million (tpm); expanded scale on right.

Proposed Research

"Blooms" of toxic dinoflagellates from several genera result in
outbreaks of paralytic shellfish poisoning (PSP), one of the more
serious of the global phenomena commonly called red tides or harmful
algal blooms. The economic, public health, and ecosystem impacts
of PSP outbreaks take a variety of forms, and include human intoxications
and death from contaminated shellfish or fish, alterations of marine
trophic structure, and death of marine mammals, fish, and seabirds.
These impacts are caused by the saxitoxins, a family of neurotoxins
produced by some dinoflagellates that accumulate in zooplankton,
shellfish, or fish during feeding.

While the chemical structure and activity of saxitoxins (STX) have
been characterized, little is known about their biosynthetic pathway
or metabolic role within the dinoflagellate. To better understand
the regulation of toxin production in dinoflagellates, our laboratory
has attempted to isolate gene(s) involved in toxin synthesis or
its regulation in Alexandrium fundyense, a toxic dinoflagellate
responsible for PSP outbreaks in the northeastern U.S. This has
been a challenging task, however, as the use of conventional genetic
tools is limited by biological and technical factors associated
with dinoflagellates. These include a lack of information on the
number of copies of ?toxin genes? and the extraordinarily high content
of DNA per cell. Our efforts to identify STX genes have focused
on the identification and cloning of differentially expressed gene
transcripts, utilizing methods that detect the presence or absence
of a gene under a particular condition. These methods have been
unsuccessful due to DNA sequence differences between our toxic strains
and the closely related non-toxic strains to which we compared them.

To circumvent this problem, we propose to employ a recently developed
method to identify genes whose expression is increased or decreased
(not just present or absent) under particular conditions. Massively
Parallel Signature Sequencing (MPSS) generates a short (17 nucleotide),
diagnostic sequence ?tag? for one million individual gene transcripts
in a cell, allowing the identification, comparison and quantification
of the full cellular complement of expressed genes. In order to
maximize the data obtained from the MPSS analysis, we will use cells
grown under nitrogen and phosphorus starvation, conditions that
decrease and increase cellular toxin content respectively. We then
should be able to identify genes for toxin production, N stress
and P stress, as sequences specific to each of these conditions
should be differentially regulated between the two samples. MPSS
analysis is extremely powerful, and will likely be used by several
laboratories at WHOI once the procedures are demonstrated on a model
system such as ours. Simply stated, no other approach currently
available will give us the number of expressed sequence tags for
a lower cost or in a more practical format. MPSS is costly, however,
and funds from the Ocean Life Institute are needed if we are to
take advantage of this extremely powerful new technology to address
several longstanding issues in harmful algal bloom ecology. Knowledge
of toxin-associated genes will help to clarify the thus far elusive
STX biosynthetic pathway in dinoflagellates and facilitate studies
on the regulation of STX production, thereby greatly augmenting
our understanding of toxic bloom formation. In addition, the STX
gene sequences will enable the design of molecular probes that unequivocally
identify only toxic cells in a complex natural assemblage of plankton,
and these are of use in academic field research as well as in commercial
and governmental shellfish toxicity monitoring programs.

Progress Report

Funding from the Ocean Life Institute has enabled us to take advantage of an extremely powerful new technology to address a longstanding issue in harmful algal bloom ecology: the identification of the genes for biosynthesis of the dinoflagellate neurotoxin saxitoxin. Production of this toxin by dinoflagellates from several genera result in outbreaks of paralytic shellfish poisoning (PSP), one of the more serious of the global phenomena commonly called red tides or harmful algal blooms. The economic, public health, and ecosystem impacts of PSP outbreaks take a variety of forms, and include human intoxications and death from contaminated shellfish or fish, alterations of marine trophic structure, and death of marine mammals, fish, and seabirds. Identification of toxin-associated genes will help to clarify the thus far elusive STX biosynthetic pathway in dinoflagellates and facilitate studies on the regulation of STX production, thereby greatly augmenting our understanding of toxic bloom formation. In addition, the STX gene sequences will enable the design of molecular probes that unequivocally identify only toxic cells in a complex natural assemblage of plankton, and these are of use in academic field research as well as in commercial and governmental shellfish toxicity monitoring programs.

Our experimental approach employed a recently developed method to identify genes whose expression is increased or decreased under particular conditions. Massively Parallel Signature Sequencing (MPSS) generates a short (17 nucleotide), diagnostic sequence ?tag? for one million individual gene transcripts in a cell, allowing the identification, comparison and quantification of the full cellular complement of expressed genes. In order to maximize the data obtained from the OLI-funded MPSS analysis, we used cells grown under nitrogen and phosphorus starvation, conditions that decrease and increase cellular toxin content respectively. The results should allow us to identify genes for toxin production, N stress and P stress, as sequences specific to each of these conditions should be differentially regulated between the two samples.

Preliminary analysis of the data provides some intriguing glimpses into dinoflagellate gene regulation. Alexandrium cells contain many more signature tags (~40,000) than do humans (~25,000) or Arabadopsis (~16,000-17,000). Expression levels of a large number of these signatures are significantly different between N- and P-limited conditions (3172 tags at p<0.001). Many signatures are unique to one condition or the other (at p<0.001); 526 signatures are found only under -P conditions and 1412 are unique to N starvation. Data for all signatures that are differentially expressed at p<0.001 are displayed graphically below. Efforts are currently underway to obtain additional sequence information via 3? RACE and EST sequencing of cDNA libraries for genes that are unique to or more highly expressed under P starvation. This includes 1265 signatures at p<0.001 and is expected to include genes associated with toxin synthesis and the P starvation response.

Originally published: February 1, 2002

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